BackgroundIn mammals, the gut microbiota has important effects on the health of their hosts. Recent research highlights that animal populations that live in captivity often differ in microbiota diversity and composition from wild populations. However, the changes that may occur when animals move to captivity remain difficult to predict and factors generating such differences are poorly understood. Here we compare the bacterial gut microbiota of wild and captive Damaraland mole-rats (Fukomys damarensis) originating from a population in the southern Kalahari Desert to characterise the changes of the gut microbiota that occur from one generation to the next generation in a long-lived, social rodent species.ResultsWe found a clear divergence in the composition of the gut microbiota of captive and wild Damaraland mole-rats. Although the dominating higher-rank bacterial taxa were the same in the two groups, captive animals had an increased ratio of relative abundance of Firmicutes to Bacteroidetes compared to wild animals. The Amplicon Sequence Variants (ASVs) that were strongly associated with wild animals were commonly members of the same bacterial families as those strongly associated with captive animals. Captive animals had much higher ASV richness compared to wild-caught animals, explained by an increased richness within the Firmicutes.ConclusionWe found that the gut microbiota of captive hosts differs substantially from the gut microbiota composition of wild hosts. The largest differences between the two groups were found in shifts in relative abundances and diversity of Firmicutes and Bacteroidetes.

BackgroundStaphylococcus aureus is a common cause of clinical mastitis (CM) in dairy cattle. Optimizing the bovine mammary gland microbiota to resist S. aureus colonization is a growing area of research. However, the details of the interbacterial interactions between S. aureus and commensal bacteria, which would be required to manipulate the microbiome to resist infection, are still unknown. This study aims to characterize changes in the bovine milk bacterial community before, during, and after S. aureus CM and to compare bacterial communities present in milk between infected and healthy quarters.MethodsWe collected quarter-level milk samples from 698 Holstein dairy cows over an entire lactation. A total of 11 quarters from 10 cows were affected by S. aureus CM and milk samples from these 10 cows (n = 583) regardless of health status were analyzed by performing 16S rRNA gene amplicon sequencing.ResultsThe milk microbiota of healthy quarters was distinguishable from that of S. aureus CM quarters two weeks before CM diagnosis via visual inspection. Microbial network analysis showed that 11 OTUs had negative associations with OTU0001 (Staphylococcus). A low diversity or dysbiotic milk microbiome did not necessarily correlate with increased inflammation. Specifically, Staphylococcus xylosus, Staphylococcus epidermidis, and Aerococcus urinaeequi were each abundant in milk from the quarters with low levels of inflammation.ConclusionOur results show that the udder microbiome is highly dynamic, yet a change in the abundance in certain bacteria can be a potential indicator of future S. aureus CM. This study has identified potential prophylactic bacterial species that could act as a barrier against S. aureus colonization and prevent future instances of S. aureus CM.

BackgroundThe critically endangered kākāpō is a flightless, nocturnal parrot endemic to Aotearoa New Zealand. Recent efforts to describe the gastrointestinal microbial community of this threatened herbivore revealed a low-diversity microbiota that is often dominated by Escherichia-Shigella bacteria. Given the importance of associated microbial communities to animal health, and increasing appreciation of their potential relevance to threatened species conservation, we sought to better understand the development of this unusual gut microbiota profile. To this end, we conducted a longitudinal analysis of faecal material collected from kākāpō chicks during the 2019 breeding season, in addition to associated nest litter material.ResultsUsing an experimental approach rarely seen in studies of threatened species microbiota, we evaluated the impact of a regular conservation practice on the developing kākāpō microbiota, namely the removal of faecal material from nests. Artificially removing chick faeces from nests had negligible impact on bacterial community diversity for either chicks or nests (p > 0.05). However, the gut microbiota did change significantly over time as chick age increased (p < 0.01), with an increasing relative abundance of Escherichia-Shigella coli over the study period and similar observations for the associated nest litter microbiota (p < 0.01). Supplementary feeding substantially altered gut bacterial diversity of kākāpō chicks (p < 0.01), characterised by a significant increase in Lactobacillus bacteria.ConclusionsOverall, chick age and hand rearing conditions had the most marked impact on faecal bacterial communities. Similarly, the surrounding nest litter microbiota changed significantly over time since a kākāpō chick was first placed in the nest, though we found no evidence that removal of faecal material influenced the bacterial communities of either litter or faecal samples. Taken together, these observations will inform ongoing conservation and management of this most enigmatic of bird species.

Parasitic infections disturb gut microbial communities beyond their natural range of variation, possibly leading to dysbiosis. Yet it remains underappreciated that most infections are accompanied by one or more co-infections and their collective impact is largely unexplored. Here we developed a framework illustrating changes to the host gut microbiome following single infections, and build on it by describing the neutral, synergistic or antagonistic impacts on microbial α- and ß-diversity expected from co-infections. We tested the framework on microbiome data from a non-human primate population co-infected with helminths and Adenovirus, and matched patterns reported in published studies to the introduced framework. In this case study, α-diversity of co-infected Malagasy mouse lemurs (Microcebus griseorufus) did not differ in comparison with that of singly infected or uninfected individuals, even though community composition captured with ß-diversity metrices changed significantly. Explicitly, we record stochastic changes in dispersion, a sign of dysbiosis, following the Anna-Karenina principle rather than deterministic shifts in the microbial gut community. From the literature review and our case study, neutral and synergistic impacts emerged as common outcomes from co-infections, wherein both shifts and dispersion of microbial communities following co-infections were often more severe than after a single infection alone, but microbial α-diversity was not universally altered. Important functions of the microbiome may also suffer from such heavily altered, though no less species-rich microbial community. Lastly, we pose the hypothesis that the reshuffling of host-associated microbial communities due to the impact of various, often coinciding parasitic infections may become a source of novel or zoonotic diseases.

BackgroundThe use of rumen microbial community (RMC) profiles to predict methane emissions has driven interest in ruminal DNA preservation and extraction protocols that can be processed cheaply while also maintaining or improving DNA quality for RMC profiling. Our standard approach for preserving rumen samples, as defined in the Global Rumen Census (GRC), requires time-consuming pre-processing steps of freeze drying and grinding prior to international transportation and DNA extraction. This impedes researchers unable to access sufficient funding or infrastructure. To circumvent these pre-processing steps, we investigated three methods of preserving rumen samples for subsequent DNA extraction, based on existing lysis buffers Tris-NaCl-EDTA-SDS (TNx2) and guanidine hydrochloride (GHx2), or 100% ethanol.ResultsRumen samples were collected via stomach intubation from 151 sheep at two time-points 2 weeks apart. Each sample was separated into four subsamples and preserved using the three preservation methods and the GRC method (n = 4 × 302). DNA was extracted and sequenced using Restriction Enzyme-Reduced Representation Sequencing to generate RMC profiles. Differences in DNA yield, quality and integrity, and sequencing metrics were observed across the methods (p < 0.0001). Ethanol exhibited poorer quality DNA (A260/A230 < 2) and more failed samples compared to the other methods. Samples preserved using the GRC method had smaller relative abundances in gram-negative genera Anaerovibrio, Bacteroides, Prevotella, Selenomonas, and Succiniclasticum, but larger relative abundances in the majority of 56 additional genera compared to TNx2 and GHx2. However, log10 relative abundances across all genera and time-points for TNx2 and GHx2 were on average consistent (R2 > 0.99) but slightly more variable compared to the GRC method. Relative abundances were moderately to highly correlated (0.68 ± 0.13) between methods for samples collected within a time-point, which was greater than the average correlation (0.17 ± 0.11) between time-points within a preservation method.ConclusionsThe two modified lysis buffers solutions (TNx2 and GHx2) proposed in this study were shown to be viable alternatives to the GRC method for RMC profiling in sheep. Use of these preservative solutions reduces cost and improves throughput associated with processing and sequencing ruminal samples. This development could significantly advance implementation of RMC profiles as a tool for breeding ruminant livestock.

BackgroundDespite well-known effects of diet on gut microbiota diversity, relatively little is known about how maternal diet quality shapes the longitudinal maturation of gut microbiota in offspring. To investigate, we fed female rats standard chow (Chow) or a western-style, high-choice cafeteria diet (Caf) prior to and during mating, gestation and lactation. At weaning (3 weeks), male and female offspring were either maintained on their mother’s diet (ChowChow, CafCaf groups) or switched to the other diet (ChowCaf, CafChow). Fecal microbial composition was assessed in dams and longitudinally in offspring at 3, 7 and 14 weeks of age.ResultsThe effect of maternal diet on maturation of offspring gut microbiota was assessed by α- and β-diversities, Deseq2/LEfSe, and SourceTracker analyses. Weanling gut microbiota composition was characterised by reduced α- and β-diversity profiles that clustered away from dams and older siblings. After weaning, offspring gut microbiota came to resemble an adult-like gut microbiota, with increased α-diversity and reduced dissimilarity of β-diversity. Similarly, Deseq2/LEfSe analyses found fewer numbers of altered operational taxonomic units (OTUs) between groups from weaning to adulthood. SourceTracker analyses indicated a greater overall contribution of Caf mothers’ microbial community (up to 20%) to that of their offspring than the contribution of Chow mothers (up to 8%). Groups maintained on the maternal diet (ChowChow, CafCaf), versus those switched to the other diet (ChowCaf, CafChow) post-weaning significantly differed from each other at 14 weeks (Permutational Multivariate Analysis of Variance), indicating interactive effects of maternal and post-weaning diet on offspring gut microbiota maturation. Nevertheless, this developmental trajectory was unaffected by sex and appeared consistent between ChowChow, CafCaf, ChowCaf and CafChow groups.ConclusionsIntroducing solid food at weaning triggered the maturation of offspring gut microbiota to an adult-like profile in rats, in line with previous human studies. Postweaning Caf diet exposure had the largest impact on offspring gut microbiota, but this was modulated by maternal diet history. An unhealthy maternal Caf diet did not alter the developmental trajectory of offspring gut microbiota towards an adult-like profile, insofar as it did not prevent the age-associated increase in α-diversity and reduction in β-diversity dissimilarity.

BackgroundInter-population variation in host-associated microbiota reflects differences in the hosts’ environments, but this characterization is typically based on studies comparing few populations. The diversity of natural habitats and captivity conditions occupied by any given host species has not been captured in these comparisons. Moreover, intraspecific variation in gut microbiota, generally attributed to diet, may also stem from differential acquisition of environmental microbes—an understudied mechanism by which host microbiomes are directly shaped by environmental microbes. To more comprehensively characterize gut microbiota in an ecologically flexible host, the ring-tailed lemur (Lemur catta; n = 209), while also investigating the role of environmental acquisition, we used 16S rRNA sequencing of lemur gut and soil microbiota sampled from up to 13 settings, eight in the wilderness of Madagascar and five in captivity in Madagascar or the U.S. Based on matched fecal and soil samples, we used microbial source tracking to examine covariation between the two types of consortia.ResultsThe diversity of lemur gut microbes varied markedly within and between settings. Microbial diversity was not consistently greater in wild than in captive lemurs, indicating that this metric is not necessarily an indicator of host habitat or environmental condition. Variation in microbial composition was inconsistent both with a single, representative gut community for wild conspecifics and with a universal ‘signal of captivity’ that homogenizes the gut consortia of captive animals. Despite the similar, commercial diets of captive lemurs on both continents, lemur gut microbiomes within Madagascar were compositionally most similar, suggesting that non-dietary factors govern some of the variability. In particular, soil microbial communities varied across geographic locations, with the few samples from different continents being the most distinct, and there was significant and context-specific covariation between gut and soil microbiota.ConclusionsAs one of the broadest, single-species investigations of primate microbiota, our study highlights that gut consortia are sensitive to multiple scales of environmental differences. This finding begs a reevaluation of the simple ‘captive vs. wild’ dichotomy. Beyond the important implications for animal care, health, and conservation, our finding that environmental acquisition may mediate aspects of host-associated consortia further expands the framework for how host-associated and environmental microbes interact across different microbial landscapes.

BackgroundBlack soldier fly (Hermetia illucens) is a promising insect species to use as a novel ingredient in fish feeds. Black soldier fly larvae consists of three major fractions, namely protein, lipid, and exoskeleton. These fractions contain bioactive compounds that can modulate the gut microbiota in fish such as antimicrobial peptides, lauric acid, and chitin. However, it is not certain how, or which fractions of black solider fly would affect gut microbiota in fish. In the present study, black soldier fly larvae were processed into three different meals (full-fat, defatted and de-chitinized) and two fractions (oil and exoskeleton), and included in diets for Atlantic salmon (Salmo salar). Atlantic salmon pre-smolts were fed with these diets in comparison with a commercial-like control diet for eight weeks to investigate the effects of insect meals and fractions on the composition and predicted metabolic capacity of gut microbiota. The gut microbiota was profiled by 16S rRNA gene sequencing, and the predicted metabolic capacities of gut microbiota were determined using genome-scale metabolic models.ResultsThe inclusion of insect meals and fractions decreased abundance of Proteobacteria and increased abundance of Firmicutes in salmon gut. The diets that contained insect chitin, i.e., insect meals or exoskeleton diets, increased abundance of chitinolytic bacteria including lactic acid bacteria and Actinomyces in salmon gut, with fish fed full-fat meal diet showing the highest abundances. The diets that contained insect lipids, i.e., insect meals and oil diets enriched Bacillaceae in fish gut. The fish fed diets containing full-fat insect meal had a unique gut microbiota composition dominated by beneficial lactic acid bacteria and Actinomyces, and showed a predicted increase in mucin degradation compared to the other diets.ConclusionsThe present results showed that the dietary inclusion of insect meals and fractions can differently modulate the composition and predicted metabolic capacity of gut microbiota in Atlantic salmon pre-smolts. The use of full-fat black soldier fly larvae meal in diets for salmon is more favorable for beneficial modulation of gut microbiota than larvae processed by separation of lipid or exoskeleton fractions.

BackgroundFish gut microbial colonisation starts during larval stage and plays an important role in host’s growth and health. To what extent first colonisation could influence the gut microbiome succession and growth in later life remains unknown. In this study, Nile tilapia embryos were incubated in two different environments, a flow-through system (FTS) and a biofloc system (BFS); hatched larvae were subsequently cultured in the systems for 14 days of feeding (dof). Fish were then transferred to one common recirculating aquaculture system (RAS1, common garden, 15–62 dof), followed by a growth trial in another RAS (RAS2, growth trial, 63–105 dof). In RAS2, fish were fed with two types of diet, differing in non-starch polysaccharide content. Our aim was to test the effect of rearing environment on the gut microbiome development, nutrient digestibility and growth performance of Nile tilapia during post-larvae stages.ResultsLarvae cultured in the BFS showed better growth and different gut microbiome, compared to FTS. After the common garden, the gut microbiome still showed differences in species composition, while body weight was similar. Long-term effects of early life rearing history on fish gut microbiome composition, nutrient digestibility, nitrogen and energy balances were not observed. Still, BFS-reared fish had more gut microbial interactions than FTS-reared fish. A temporal effect was observed in gut microbiome succession during fish development, although a distinct number of core microbiome remained present throughout the experimental period.ConclusionOur results indicated that the legacy effect of first microbial colonisation of the fish gut gradually disappeared during host development, with no differences in gut microbiome composition and growth performance observed in later life after culture in a common environment. However, early life exposure of larvae to biofloc consistently increased the microbial interactions in the gut of juvenile Nile tilapia and might possibly benefit gut health.

BackgroundRumen microorganisms carry antimicrobial resistance genes which pose a threaten to animals and humans in a One Health context. In order to tackle the emergence of antimicrobial resistance it is vital to understand how they appear, their relationship with the host, how they behave as a whole in the ruminal ecosystem or how they spread to the environment or humans. We sequenced ruminal samples from 416 Holstein dairy cows in 14 Spanish farms using nanopore technology, to uncover the presence of resistance genes and their potential effect on human, animal and environmental health.ResultsWe found 998 antimicrobial resistance genes (ARGs) in the cow rumen and studied the 25 most prevalent genes in the 14 dairy cattle farms. The most abundant ARGs were related to the use of antibiotics to treat mastitis, metritis and lameness, the most common diseases in dairy cattle. The relative abundance (RA) of bacteriophages was positively correlated to the ARGs RA.The heritability of the RA of the more abundant ARGs ranged between 0.10 (mupA) and 0.49 (tetW), similar to the heritability of the RA of microbes that carried those ARGs. Even though these genes are carried by the microorganisms, the host is partially controlling their RA by having a more suitable rumen pH, folds, or other physiological traits that promote the growth of those microorganisms.ConclusionsWe were able to determine the most prevalent ARGs (macB, msbA, parY, rpoB2, tetQ and TaeA) in the ruminal bacteria ecosystem. The rumen is a reservoir of ARGs, and strategies to reduce the ARG load from livestock must be pursued.